Word count: 7316 One sentence summary:Movement of sub-synaptic vesicles rich in LAT is constrained by protein microclusters at T cell immune synapses, dependent on LAT residues important for its binding to SLP-76 and consistent with a role for vesicular LAT in T cell signal transduction.

T-cell receptors (TCRs) undergo microclustering and supramolecular activation cluster formation at the immunological synapse (IS) during conjugation between T cells and antigen-presenting cells (APCs) (5,21,35). Microclustering can in turn activate GTP-binding proteins, protein kinases, phosphatases, and the phosphorylation of adaptor proteins. CD4-and CD8-p56lck activation leads to immunoreceptor tyrosinebased activation motif phosphorylation on the CD3 and TCR chains, the recruitment of ZAP-70 (zeta-associated proteintyrosine kinase of 70 kDa), and the activation of TEC kinases ITK/RLK (interleukin-2-inducible/resting lymphocyte kinase) (1,(31)(32)(33)41). Adaptor proteins possess binding sites and domains needed for complex-complex formation (31,32,41). Immune cell-specific adaptors include LAT (linker for activation of T cells), GADS (Grb-2-like adaptor downstream of Shc), SLP-76 (SH2 domain-containing leukocyte protein of 76 kDa), ADAP (adhesion-and degranulation-promoting adaptor protein; previously known as FYN T-binding protein/SLP-76-associated protein [FYB/SLAP]), and SKAP-55 (Src kinase-associated phosphoprotein of 55 kDa; also known as SCAP1) (3,23,24,26,31,32,(40)(41)(42). Phosphorylation of LAT recruits phospholipase C␥1, Grb-2, and GADS-SLP-76 and induces Ca 2ϩ mobilization and cytokine transcription (32,33,41,44).Binding of leukocyte function-associated antigen 1 (LFA-1; also known as CD11/CD18 or ␣ L ␤ 2 ) to intercellular adhesion molecules 1 and 2 (ICAM-1 and -2) on APCs mediates T-cell-APC conjugation (5,8,11,21,37). Following initial adhesion, TCR/CD3 ligation induces signals (i.e., "inside-out signaling") that further activate integrin adhesion (2,5,8,11,37). Conversion of LFA-1 to intermediate-or higher-affinity forms involves changes in conformation and receptor clustering (11,37). Multiple signaling proteins mediate this process. They include the GTP-binding protein Rap-1, its ligand RapL (regulator of cell adhesion and polarization enriched in lymphoid tissues), RIAM (Rap1-GTP-interacting adaptor molecule), the guanine nucleotide exchange factor Vav-1, and the adaptors ADAP,10,13,15,16,17,18,20,22,27,28,34). The protein-tyrosine kinase ZAP-70 phosphorylates YESP sites in SLP-76, which allows binding to the Src homology 2 (SH2) domain of Vav-1 (29), while the SH2 domain of SLP-76 binds to two YDDV sites in ADAP (30, 39). T-cell lines lacking SLP-76 show impaired superantigen-induced conjugation (44). ADAP is an immune cell-specific adaptor with a unique N-terminal region, a proline-rich region, a canonical and a noncanonical SH3 domain, one Ena/VASP homology 1 (EVH1) binding domain, and two putative nuclear localization motifs (3,4,19,26,31). ADAP is preferentially phosphorylated by the Src kinase p59fynT (4,25) and can cooperate with p59 fynT and SLP-76 in amplifying TCR-induced interleukin-2 (IL-2) transcription (30). The adaptor can up-regulate integrin-mediated adhesion in certain basophilic cell lines (9), while ADAP Ϫ/Ϫ T cells show profound defects in ␤1 and ␤2 integrin clusterin...

CTLA-4 is a co-receptor that modulates the threshold of T cell activation and autoimmunity. We previously showed that CTLA-4 reverses the TCR-mediated stop signal needed for T cell/APC interactions [Schneider et al., Science 2006, 313: 1972. In this study, using a different T cell system, we show that CTLA-4 expression changed the behavior of T8.1 T cells by reducing the contact time between T cell and APC, preventing re-inforced contacts, and reducing the contact area at the immunological synapse. This led to a major reduction in Ca 2+ influx/mobilization and interleukin-2 production. Further, anti-CD3/CTLA-4 increased T cell motility on antibody-coated glass slides, concurrent with an abrogation of ZAP70 microcluster formation. Our findings further support a role for CTLA-4 in limiting the interaction between T cell and APC that is needed for optimal activation.

The multipotent cytokine granulocyte macrophage-colony stimulating factor (GM-CSF) is involved in particular in the physiological response to infection and in inflammatory responses. GM-CSF is produced by many cell types, including T lymphocytes responding to T-cell receptor activation and mantle zone B lymphocytes. B-cell receptor and T-cell receptor activation generates two major signals: an increase in intracellular Ca(2+) concentration and a protein kinase cascade. Previous studies have shown that the Ca(2+)/calmodulin-dependent phosphatase calcineurin mediates stimulation of GM-CSF transcription in response to Ca(2+). In this study, we show that Ca(2+) signaling also regulates GM-CSF transcription negatively through Ca(2+)/calmodulin-dependent kinase II (CaMK II) phosphorylation of serines in the autoinhibitory domain for DNA binding of the transcription factor Ets1. Wild-type Ets1 negatively affects GM-CSF transcription on Ca(2+) stimulation in the presence of cyclosporin A, which inhibits calcineurin. Conversely, Ets1 with mutated CaMK II target serines showed an increase in transactivation of the GM-CSF promoter/enhancer. Moreover, constitutively active CaMK II inhibited transactivation of GM-CSF by wild-type Ets1 but not by Ets1 with mutated CaMK II sites. Mutation of CaMK II target serines in Ets1 also relieves inhibition of cooperative transactivation of GM-CSF with the Runx1/AML1 transcription factor. In addition, the Ca(2+)-dependent phosphorylation of Ets1 reduces the binding of Ets1 to the GM-CSF promoter in vivo.

Infectious bursal disease virus (IBDV) is a bi-segmented double-strand RNA (dsRNA) virus of the family. While IBDV genomic dsRNA lacks a 5' cap, the means by which the uncapped IBDV genomic RNA is translated effectively is unknown. In this study, we describe a cap-independent pathway of translation initiation of IBDV uncapped RNA that relies on VP1 and VP3. We show that neither purified IBDV genomic dsRNA nor the uncapped viral plus-sense RNA transcripts was directly translated and rescued into infectious viruses in host cells. This defect in translation of the uncapped IBDV genomic dsRNA was rescued by-supplementation of the viral proteins VP1 and VP3, which was dependent on both the intact polymerase activity of VP1 and the dsRNA binding activity of VP3. Deletion analysis showed that both 5' - and 3' -UTRs of IBDV dsRNA were essential for the VP1/VP3-dependent translation initiation. Significantly, VP1 and VP3 could also mediate the recovery of infectious IBDV from the authentic minus-sense strand of IBDV dsRNA. Moreover, down-regulation or inhibition of the cap-binding protein eIF4E did not decrease, but rather enhanced the VP1/VP3-mediated translation of the uncapped IBDV RNA. Collectively, our findings for the first time reveal that VP1 and VP3 compensate for the deficiency of 5' cap and replace eIF4E to confer upon the uncapped IBDV RNA the ability to be translated and rescued into infectious viruses.A key point of control for virus replication is the viral translation initiation. The current study shows that the uncapped IBDV RNA cannot be translated into viral proteins directly by host translation machinery, and is thus noninfectious. Our results constitute the first direct experimental evidence that the VP1 and VP3 are required and sufficient to initiate translation of uncapped IBDV genomic RNA by acting as a substitute of cap and replacing the cap-binding protein eIF4E. Significantly, the VP1/VP3 mediate the recovery of infectious IBDV not only from the plus-sense but also from the minus-sense strand of the IBDV dsRNA. These findings provide not only new insights into the molecular mechanisms of the life cycle of IBDV, but also a new tool for an alternative strategy for the recovery of IBDV from both the plus- and the minus-sense strand of the viral genomic dsRNA.

Acute myeloid leukemia 1 (AML1), also denoted Runx1, is a transcription factor essential for hematopoiesis, and the AML1 gene is the most common target of chromosomal translocations in human leukemias. AML1 binds to sequences present in the regulatory regions of a number of hematopoiesis-specific genes, including certain cytokines such as granulocyte macrophage colony-stimulating factor (GM-CSF) up-regulated after T cell receptor stimulation. Here we show that both subunits of the Ca 2؉ / calmodulin-dependent protein phosphatase calcineurin (CN), which is activated upon T cell receptor stimulation, interact directly with the N-terminal runt homology domain-containing part of AML1. The regulatory CN subunit binds AML1 with a higher affinity and in addition also interacts with the isolated runt homology domain. The related Runx2 transcription factor, which is essential for bone formation, also interacts with CN. A constitutively active derivative of CN is shown to activate synergistically the GM-CSF promoter/enhancer together with AML1 or Runx2. We also provide evidence that relief of the negative effect of the AML1 sites is important for Ca 2؉ activation of the GM-CSF promoter/enhancer and that AML1 overexpression increases this Ca 2؉ activation. Both subunits of CN interact with AML1 in coimmunoprecipitation analyses, and confocal microscopy analysis of cells expressing fluorescence-tagged protein derivatives shows that CN can be recruited to the nucleus by AML1 in vivo. Mutant analysis of the GM-CSF promoter shows that the Ets1 binding site of the promoter is essential for the synergy between AML1 and CN in Jurkat T cells. Analysis of the effects of inhibitors of the protein kinase glycogen synthase kinase-3␤ and in vitro phosphorylation/dephosphorylation analysis of Ets1 suggest that glycogen synthase kinase-3␤-phosphorylated Ets1 is a target of AML1-recruited CN phosphatase at the GM-CSF promoter.

SummaryWhile immune cell adaptors regulate proximal T cell signaling, direct regulation of the nuclear pore complex (NPC) has not been reported. NPC has cytoplasmic filaments composed of RanGAP1 and RanBP2 with the potential to interact with cytoplasmic mediators. Here, we show that the immune cell adaptor SLP-76 binds directly to SUMO-RanGAP1 of cytoplasmic fibrils of the NPC, and that this interaction is needed for optimal NFATc1 and NF-κB p65 nuclear entry in T cells. Transmission electron microscopy showed anti-SLP-76 cytoplasmic labeling of the majority of NPCs in anti-CD3 activated T cells. Further, SUMO-RanGAP1 bound to the N-terminal lysine 56 of SLP-76 where the interaction was needed for optimal RanGAP1-NPC localization and GAP exchange activity. While the SLP-76-RanGAP1 (K56E) mutant had no effect on proximal signaling, it impaired NF-ATc1 and p65/RelA nuclear entry and in vivo responses to OVA peptide. Overall, we have identified SLP-76 as a direct regulator of nuclear pore function in T cells.

While the entry of infectious bursal disease virus (IBDV) is initiated by the binding of the virus to the two major receptors integrin and HSP90, the signaling events after receptor binding and how they contribute to virus entry remain elusive. We show here that IBDV activates c-Src by inducing the phosphorylation of the Y416 residue in c-Src both in DF-1 chicken fibroblasts and in vivo in the bursa of Fabricius from specific-pathogen-free (SPF) chickens. Importantly, inactivated IBDV fails to stimulate c-Src Y416 phosphorylation, and a very virulent IBDV strain induces a much higher level of c-Src Y416 phosphorylation than does an attenuated strain. Inhibition of c-Src activation by an Src kinase inhibitor or expression of a c-Src dominant negative mutant results in a significant decrease in the internalization of IBDV but has little effect on virus adhesion. Furthermore, short hairpin RNA (shRNA) downregulation of integrin, either the ␣4 or ␤1 subunit, but not HSP90 remarkably attenuates IBDV-induced c-Src Y416 phosphorylation, resulting in a decrease in IBDV internalization but not virus adhesion. Moreover, interestingly, inhibition of either c-Src downstream of the phosphatidylinositol 3-kinase (PI3K)/Akt-RhoA signaling cascade or actin rearrangement leads to a significant decrease in IBDV internalization irrespective of the IBDV-induced high levels of c-Src phosphorylation. Cumulatively, our results suggest a novel feed-forward model whereby IBDV activates c-Src for benefiting its cell entry via an integrin-mediated pathway by the activation of downstream PI3K/Akt-RhoA signaling and cytoskeleton actin rearrangement.IMPORTANCE While IBDV-caused immunosuppression is highly related to viral invasion, the molecular basis of the cellular entry of IBDV remains elusive. In this study, we demonstrate that IBDV activates c-Src by inducing the phosphorylation of the Y416 residue in c-Src to promote virus internalization but not virus adhesion. The ability to induce the level of c-Src Y416 phosphorylation correlates with the pathogenicity of an IBDV strain. IBDV-induced c-Src Y416 activation is ␣4␤1 integrin but not HSP90 dependent and involves the activation of the downstream PI3K/Akt-RhoA GTPase-actin rearrangement cascade. Thus, our findings provide new insights into the IBDV infection process and the potential for c-Src as a candidate target for the development of IBDV therapeutic drugs.